JP3030982B2 - Novel GL-7ACA acylase - Google Patents

Abstract

A GL-7ACA acylase having the following characteristics: (a) has ability to catalyze the enzymatic conversion of glutaryl 7-ACA, adipyl 7-ACA, and succinyl 7-ACA, into 7-aminocephalosporanic acid, (b) has a molecular weight of 70,000 dalton (SDS-PAGE) and (c) has N-terminal amino acid sequence thereof : Gln-Ser-Glu-Gln-Glu-Lys-Ala-Glu-Glu-. A process for producing GL-7ACA acylase is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel GL-7ACA.
More specifically, the present invention relates to a novel GL-7A derived from Bacilluslaterosporus J1 with respect to acylase (hereinafter, referred to as “GL-7ACA acylase J1”).
The present invention relates to a CA acylase, a DNA encoding the same, an expression vector containing the DNA, a microorganism transformed with the expression vector, and production of GL-7ACA by culturing the transformant.

[0002]

2. Description of the Related Art GL-7ACA acylase is a 7-
Hydrolysis of (4-carboxybutanamide) -3-acetoxymethyl-3-cephem-4-carboxylic acid (GL-7ACA) to give 7-aminocephalosporanic acid (7-ACA)
A common name for enzymes that have in common that they can be converted to. Conventionally, 7-ACA has been produced by a chemical conversion method of cephalosporin such as an imino ether method or a nitrosyl chloride method. However, the starting material for penicillins, another family of β-lactam antibiotics, is 6-.
Since an enzymatic conversion method similar to the production of aminopenicillanic acid (6-APA) has been successfully adopted, alternatives utilizing enzymatic conversion have long been sought to reduce costs. In the course of such efforts, a two-step enzymatic conversion method using D-amino acid oxidase and glutaryl 7ACA (GL-7ACA) acylase was devised. This method is industrialized as a method in which enzymatic oxidation is replaced by chemical oxidation.

[0003] Pseudomonas sp. Strain GK16 is GL-7.
Produces ACA acylase and reveals part of the nucleotide sequence of the acylase gene
[Journal of Bacteriology 163, 122
Pp. 2-1228 (1985)].

[0004]

Means for Solving the Problems The inventors of the present invention provide:
Intensive research was conducted for a new GL-7ACA acylase. As a result, the present inventors have found a novel and characteristic GL-7ACA acylase J1 in a culture of a newly isolated microorganism, Bacillus laterosporous J1,
An industrial production method of this enzyme was established. GL- of the present invention
7ACA acylase J1 is characterized by higher thermostability and a completely different structure from known GL-7ACA acylases. Other features of this GL-7ACA acylase will become apparent from the description below.

[0005] A strain named Bacillus laterosporus J1, which is a GL-7ACA acylase-producing bacterium, is newly isolated from a soil sample collected in Iwate Prefecture.

[0006] Bacillus laterosporous J1 has the following morphological and physiological characteristics and is available from the Barges Manual of Systematic Bacteriology.
(Vol. 1) and identified as Bacillus laterosporous. For this classification study, the method described in the Barges Manual is mainly used.

[0007] 1. Morphological characteristics The morphological observation of the J1 strain was performed by trypticase soy broth.
Light microscopy was performed on cells cultured at 37 ° C. (BBL, USA).

The J1 strain was a gram-positive, motile bacterium. The bacterial morphology was rod-shaped. Table 1 shows the results.

[0009]

[Table 1]

[0010] 2. Physiological characteristics Tables 2 and 3 summarize the physiological characteristics of the J1 strain. The J1 strain was positive for oxidase, positive for catalase, and negative for the OF test. Gelatin was not liquefied and esculin hydrolysis was negative. Nitrate is reduced, N ₂ gas is generated. Ribose, sucrose, trehalose and xylose were not utilized. Lysine and ornithine were not decarboxylated and arginine was not hydrolyzed. The indole test was negative. The Voges-Proskaer test was negative.

[0011]

[Table 2]

[Table 3]

The novel GL-7ACA acylase of the present invention has the following properties.

That is, the novel GL-7ACA of the present invention
The acylase has (a) a catalytic ability for enzymatic conversion of glutaryl 7-ACA, adipyl 7-ACA and succinyl 7-ACA to 7-aminocephalosporanic acid, and (b)
It has a molecular weight of 70,000 daltons (SDS-PAGE), and (c) its N-terminal amino acid sequence is Gln-Ser-Gl.
u-Gln-Glu-Lys-Ala-Glu-Glu.

[0014] The novel GL-7ACA acylase of the present invention can be prepared by recombinant DNA technology, polypeptide synthesis and the like.

That is, the novel GL-7ACA acylase is obtained by culturing a host bacterium transformed with an expression vector containing a DNA encoding the amino acid sequence of the novel GL-7ACA acylase in a medium, and removing the novel GL-7ACA acylase from the culture. It can be prepared by collecting -7ACA.

The details of this process are described in more detail below.

Examples of the host cell include microorganisms (eg, bacteria (eg, Escherichia coli, Bacillus subtilis), yeasts (eg, Saccharomyces
Cerevisiae), animal cell lines and cultured plant cells]. Preferred examples of the microorganism include bacteria, particularly strains belonging to the genus Escherichia (eg, E. coli JM109A).
TCC53323, E.C. Cori HB101ATCC336
94; Cori HB101-16FERM BP-18
72; Coli 294ATCC 31446), or a strain belonging to the genus Bacillus (eg, Bacillus subtilis ISW1214), a yeast, particularly a strain belonging to the genus Saccharomyces (eg, Saccharomyces cerevisiae A)
H22) and animal cell lines (eg, mouse L929 cells, Chinese hamster ovary (CHO) cells, etc.).

When a bacterium, particularly Escherichia coli or Bacillus subtilis, is used as a host cell, the expression vector usually contains at least a promoter, an initiation codon, and the novel GL-7AC.
It consists of a DNA encoding the amino acid sequence of A acylase, a stop codon, a terminator region, and a replicable unit. When yeast or animal cells are used as a host, the expression vector contains at least a promoter, an initiation codon, a signal peptide and the novel GL-
DNA encoding the amino acid sequence of 7ACA acylase
And a stop codon, preferably an enhancer sequence, 5 ′ of the novel GL-7ACA acylase.
-And 3'-noncoding regions, splicing junctions, polyadenylation sites and replicable units can also be inserted into the expression vector.

The promoter for expressing the novel GL-7ACA acylase in bacteria is a promoter and a Shine-Dalgarno (SD) sequence (eg, AAGG
Etc.). Preferred promoters for bacterial expression include conventional promoters (eg, the PL and trp promoters for E. coli) and the promoter of the GL-7ACA acylase J1 chromosomal gene. The new GL in yeast
Examples of a promoter for expression of -7ACA acylase include S. cerevisiae. TRP1 gene, ADHI or ADH II gene and acid phosphatase (PHO5) for S. cerevisiae
Gene promoter in mammalian cells
As promoters for -7ACA acylase expression, SV40 early or late promoter, HTLV-
LTR promoter, mouse metallothionein I (MM
T) promoter, vaccinia promoter and the like.

Preferred initiation codons include methionine codon (ATG).

Examples of the signal peptide include signal peptides of other commonly used enzymes (natural t-PA signal peptide, natural plasminogen signal peptide) and the like.

Signal peptide or said novel GL-7A
DNA encoding the amino acid sequence of CA acylase is
Partial or total synthesis of DNA using a DNA synthesizer and / or a transformant (for example, E. coli JM1)
09 (pACYJ1-3) FERM BP-3105) (for example, a restriction enzyme) which is inserted into a suitable vector (for example, pACYJ1-3) and encodes the novel GL-7ACA acylase. , Alkaline phosphatase, polynucleotide kinase, DNA ligase, DNA polymerase, etc.).

Examples of the stop codon include conventional ones (for example, TAG, TGA, etc.).

The terminator region includes a natural or synthetic terminator (eg, a GL-7ACA acylase J1 chromosome gene terminator, a synthetic fd phage terminator, etc.).

A replicable unit is a DNA compound capable of replicating the entire DNA sequence belonging to itself in a host cell,
Examples include natural plasmids, artificially decorated plasmids
(For example, a DNA fragment prepared from a natural plasmid) and a synthetic plasmid. Preferred examples of the plasmid include plasmid pBR322 or an artificial ornament thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) for use in E. coli. Then, yeast 2μ plasmid or yeast chromosomal DNA is used for yeast, and plasmid pRSVn is used.
eoATCC37198, plasmid pSV2dhfr AT
CC37145, plasmid pdBPV-MMTneoAT
CC37224, plasmid pSV2neoATCC371
49 can be mentioned for mammalian cells, respectively.

Examples of the enhancer sequence include the enhancer sequence of SV40 (72 bp).

Examples of the polyadenylation site include the polyadenylation site of SV40.

As a splicing junction,
SV40 splicing junctions.

The promoter, start codon, DNA encoding the amino acid sequence of the novel GL-7ACA acylase, stop codon and terminator region are continuously linked to an appropriate replicable unit (plasmid) and optionally an appropriate DNA fragment. The expression vector can be obtained by using a linker (eg, a linker, another restriction site, etc.) to form a circular interconnection by a conventional method (eg, digestion with a restriction enzyme, ligation using T4 DNA ligase). When a mammalian cell is used as a host cell, an enhancer sequence, a promoter, a novel GL-7A
5'-noncoding region of cDNA of CA acylase, initiation codon, DNA encoding signal peptide and amino acid sequence of novel GL-7ACA acylase, stop codon, 3'-noncoding region of cDNA of novel GL-7ACA acylase, splicing Junction and polyadenylation sites can be consecutively interconnected in a circular fashion with the appropriate replicable units by the methods described above.

A host cell can be transformed with the expression vector. Transformation (or transfection)
Conventional methods (eg, the Kushuner method in E. coli,
Transformants can be obtained by the calcium phosphate method in mammalian cells, microinjection, etc.).

In the process of the present invention, a novel GL-7ACA
In order to produce acylase, the transformant containing the expression vector thus obtained is cultured in a medium.

The nutrient medium contains a carbon source (eg, glucose, glycerin, mannitol, fructose, lactose, etc.) and an inorganic or organic nitrogen source (eg, ammonium sulfate, ammonium chloride, casein hydrolyzate,
Yeast extract, polypeptone, bactotripton, meat extract, etc.). If desired, the medium may contain other nutrient sources such as inorganic salts (eg, sodium or potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, vitamin B ₁ ), antibiotics (For example, ampicillin, kanamycin) and the like. For culture of mammalian cells, Dulbecco's Modified Eagle's Minimal Medium supplemented with fetal calf serum and antibiotics is frequently used.

Culture of the transformant (including the transfectant) is usually performed at pH 5.5 to 8.5 (preferably at pH 5.5).
7 to 7.5), 18 to 40 ° C (preferably 25 to 38 ° C)
For 5 to 50 hours.

When the novel GL-7ACA acylase thus produced is present in the culture solution, culture filtrate (supernatant), the culture is filtered or centrifuged. From the culture filtrate, the novel GL-7ACA acylase can be obtained by a conventional method generally used for purification and isolation of a natural or synthetic protein.
(For example, dialysis, gel filtration, affinity column chromatography using an anti-GL-7ACA acylase monoclonal antibody, column chromatography using an appropriate adsorbent, high performance liquid chromatography, etc.). When the produced novel GL-7ACA acylase is present in the periplasm and cytoplasm of the cultured transformant, the cells are collected by filtration or centrifugation, and their cell walls and / or cell membranes are treated with, for example, ultrasound and / or lysozyme. To obtain debris (cell debris). The debris is dissolved in an appropriate aqueous solution (for example, an 8 M aqueous urea solution, a 6 M aqueous guanidium salt solution). From this solution, a novel GL-7ACA acylase can be purified by a conventional method as described above.

The novel GL-7AC produced in E. coli
When it is necessary to regenerate (refold) A acylase, this can be performed by a conventional method.

The invention further relates to the formula:

Embedded image

Wherein R ¹ represents acetoxy, hydroxy or hydrogen; R ² represents carboxy (C ₁ -C ₆ ) alkanoyl or D-glutamyl; Contacting with a culture of a host cell transformed with an expression vector comprising DNA encoding -7ACA acylase or a processed product thereof.

Embedded image

(Wherein R ¹ has the same meaning as described above) or a salt thereof.

The carboxy (C ₁ -C ₆ ) alkanoyl represented by R ² includes glutaryl, succinyl, adipyl and the like.

Preferred salts of compounds (I) and (II) are alkali metal salts (eg, sodium and potassium salts).

When the GL-7ACA acylase activity is present in the transformed cells, the following preparations can be exemplified as processed products of the culture.

(1) Live cells; those separated from the culture by a conventional method such as filtration and centrifugation; (2) Dry cells; obtained by drying the above-mentioned live cells by a conventional method such as freeze-drying and vacuum drying (3) Cell-free extract; living cells or dried cells are subjected to a conventional method (eg, autolysis of cells using an organic solvent, disruption of cells with alumina, sea sand, etc., or ultrasonication of cells) (4) Enzyme solution; obtained by purifying or partially purifying the cell-free extract by a conventional method (eg, column chromatography); (5) Immobilized cells or immobilized cells Immobilized enzyme; prepared by immobilizing the above cells or enzymes by a conventional method (eg, acrylamide, glass beads, ion exchange resin, etc.).

When GL-7ACA acylase activity is present in the culture cell culture filtrate, the culture filtrate (supernatant), enzyme solution, immobilized cells or immobilized enzyme may be exemplified as a processed product of the culture. it can.

The reaction comprising contacting compound (II) with the enzyme can be carried out in an aqueous medium such as water or a buffer. That is, the reaction is usually carried out by dissolving or suspending the culture or a processed product thereof in an aqueous medium such as water or a buffer containing compound (II).

The preferred pH of the reaction mixture, the concentration of compound (II), the reaction time and the reaction temperature may vary depending on the nature of the culture to be used or the processed product thereof.
Generally, the reaction is carried out at pH 6-9, preferably pH 7-9,
It is carried out at 5 to 50 ° C, preferably 20 to 45 ° C, for 2 to 50 hours.

The concentration of compound (II) as a substrate in the reaction mixture is preferably selected within the range of 1 to 100 mg / ml.

The compound (I) thus produced can be purified and isolated from the reaction mixture by a conventional method.

In the following examples, the starting materials such as plasmids, restriction enzymes, enzymes such as T4 DNA ligase and other materials are commercially available. Procedures employed for DNA cloning, transformation of host cells, culture of transformants, recovery of novel GL-7ACA acylase from culture broth, and the like are well known in the art and should be appropriately adopted from the literature. Can be.

[Examples] The following examples are provided to illustrate the present invention, but the present invention is not limited thereto.

Example 1 GL-7ACA reed of Bacillus laterosporous J1
Isolation of the gene encoding the lase 1.1. B. Preparation of chromosomal DNA of Laterosporous J1
Ltd. Harris - Warwick (Harris-Warrick) et al's method (Pro
c. Natl. Acad. Sci. USA. 72 : 2207-22
11, 1975). Prepare the chromosomal DNA of Laterosporous J1. B. Laterosporous J1
Was cultured on bouillon agar (Eiken Chemical) at 30 ° C. for 40 hours, suspended in physiological saline, and collected by centrifugation.
Wash once with 50 mM Tris-HCl (pH 8) containing 1 mM EDTA. About 9 g (wet weight) of the obtained cell pellet was
Suspended in 20 ml of 50 mM Tris-HCl (pH 8) containing sucrose and 1 mM EDTA, lysozyme 25 mg
For 30 minutes at 37 ° C. In addition, 50 ml of 100 mM EDTA (pH 9.6) -1% lauroyl sarcosinate and 5 mg / ml pronase E10
Add ml and incubate the resulting mixture at 50 ° C. for 2 hours. After addition of 1.25 g of CsCl per ml of lysate, an equilibrium density gradient centrifugation is performed. After centrifugation, the chromosomal DNA fractions were pooled and 10 mM containing 1 mM EDTA.
Dialyze against Tris-HCl (pH 8) (TE buffer).

1.2. B. Geno of Laterosporous J1
B. Construction of DNA library 300 μg of chromosomal DNA of Laterosporous J1
Was partially cleaved with 3.75 units of the restriction enzyme Sau3AI, and the obtained DNA fragment was subjected to SRP28 rotor (Beckman, USA) at 26000 rpm for 10 to 40% for 20 minutes.
Subject to sucrose density gradient centrifugation. Average size 7-9
The DNA fractions in kilobases (kb) are pooled, the DNA is collected by ethanol precipitation and dissolved in TE buffer. Plasmid vector pHSG298 DNA (Takara Shuzo) (20 μg)
Is cut with BamHI (Takara Shuzo), followed by phenol extraction and ethanol precipitation. DNA was added to 10 mM ED
It is dissolved in 200 μl of 10 mM Tris-HCl (pH 8) containing TA and incubated with 1 unit of bacterial alkaline phosphatase (Takara Shuzo) at 37 ° C. for 20 minutes. The reaction mixture is treated by phenol extraction and ethanol precipitation,
Dissolve in 40 μl of buffer. Sau3AI partially truncated chromosomal DNA fragment (20 μg) and linearized dephosphorylated pHSG2
985 μg is ligated with 500 units of T4 DNA ligase (Takara Shuzo) at 12 ° C. for 16 hours. Using the ligation mixture, E. coli JM109 (Toyobo) is transformed. Transformation is performed using O. Hanahan's method (J. Mol. Bi
ol. 166 , 557-580 (1983)). Unless otherwise indicated, all transformations described below use the Hanahan method. 20 μg of transformant
Selection on LM agar containing / ml kanamycin.
The number of transformants is 2,000.

1.3. GL-7ACA acylase gene
Selection of clones containing plasmid containing GL-7ACA acylase gene Genome DN of Laterosporous J1
From the A library, select by the following HPLC assay.

A colony of the transformant was picked up and 1 mM
1 ml of 2% broth (Eiken Chemical) with isopropylthio-β-D-galactoside (IPTG; Sigma Chemical, USA)
Incubate at 30 ° C overnight. Cells are collected by centrifugation and the resulting cell pellet is used for the assay. 100
The reaction mixture (20 μl) containing mM phosphate buffer (pH 8), 2 mg GL-7ACA and the cell pellet is mixed well and incubated at 37 ° C. for 10 minutes. The reaction is stopped by adding 200 μl of 4% acetic acid. The sample was applied to a column of inertosyl ODS-2 (4.6 mm × 150 mm) (Gascro Industry), and 0.567 g / l of Na ₂ HPO ₄ , 0.3
Elution is carried out with 6 g / l KH ₂ PO ₄ and 2-4% methanol. 7ACA is detected by absorption at 254 nm.

1.4. GL-7ACA acylase
Subcloning of the gene to be cloned (see FIGS. 1 and 2), a cleared lysate method (Clewell and Helin)
ski: Proc. Natl. Acad. Sci. USA, 62 , 115
9-1166 (1969)), plasmid DNA is extracted from positive clones. This is pACYJ1
Name it -1. The size of the inserted DNA fragment is estimated to be about 8.6 kb by agarose gel electrophoresis. The recombinant plasmid pACYJ1-1 DNA (10 μg) was
I and Eco47 III (Takara Shuzo), the resulting insert DNA fragment (2.6 kb) was separated by agarose gel electrophoresis, eluted from the gel by electrophoresis, treated by phenol extraction and ethanol precipitation, and treated with TE. Dissolve in buffer. The DNA fragment (1 μg) of this insert was
Using units of T4 DNA ligase, KpnI and Hin
pHSG298DN which was linearized by digestion with cII (Takara Shuzo) and isolated in the same manner as the inserted fragment of pACYJ-1.
A1 μg. Using this ligation mixture, E. coli JM109 is transformed. Tripton (Difco) 1
%, Yeast extract (Difco) 0.5%, sodium chloride 1
Transformants were selected on LM agar plates containing 0 mM, 10 mM magnesium sulfate, 1.5% agar and 20 μg / ml kanamycin, to eliminate β-galactosidase activity, insert size and GL-7ACA acylase activity. Confirm by existence. GL-7ACA acylase activity is measured by the HPLC method as described in Example 1.3. Plasmid DNA is extracted from one of the recombinant strains by the clarified lysate method. This is pACYJ
Name it 1-3.

Example 2 GL-7ACA reed of Bacillus laterosporous J1
Determination of the nucleotide sequence of the gene encoding the enzyme

2.1. Determination of Nucleotide Sequence Restriction enzyme mapping of the insert of pCPA14-169 was performed using restriction enzymes BalI, BglII, ClaI, DraI, Eco.
RV, HpaI, NcoI, PstI, and XhoI (all manufactured by Takara Shuzo) are used. D cut with an appropriate restriction enzyme
The NA fragment is subcloned into an M13 phage vector and used for nucleotide sequence determination. Nucleotide sequences were determined using the M13 sequencing kit (Toyobo) using the dideoxy chain termination method (Sanger et al., Proc.
Natl. Acad. Sci. USA, 74 , 5463-5467
(1977)). The enzyme used is T7
It is a DNA polymerase (sequenase), and 7-deaza dGTP and dITP are also used individually as nucleotide analogs. To determine the sequence located at the center of the long DNA fragment, one of the restriction enzyme-cleaved DNA fragments was
Treated with al31, recloned into M13 phage vector and used for sequencing. Gel electrophoresis was performed at 80
Using 7 cm urea-containing polyacrylamide gel containing 7 M urea,
Perform at 200V for 5 or 13 hours. The nucleotide sequence of the inserted fragment of pACYJ1-3 is shown in FIG. 4, FIG. 5, FIG. 6, and FIG. One open reading frame of 1902 bp is recognized. From the following two results, this open reading frame is indicated by B.I. Laterosporous J1 GL-7A
It is confirmed that the gene encodes CA acylase. 1) The amino terminal sequence of GL-7ACA acylase J1 as determined by gas phase sequencing (described in detail in the following Examples) is identical to the amino acid sequence between codons 1 and 9. This indicates that processing occurs where the N-terminus, which is 27 amino acid residues long, is excised. 2) The molecular weight of 70,000 estimated by SDS-polyacrylamide gel electrophoresis is in good agreement with the molecular weight of 69,227 calculated from the predicted sequence of the protein processed for the open reading frame.
No similarity was observed between the amino acid sequence of GL-7ACA acylase J1 and the amino acid sequences of other known cephalosporin acylases.

Example 3 (see FIG. 3 ) GL-7ACA in Bacillus subtilis (Bacillus subtilis)
10 μg of the recombinant recombination plasmid pACYJ1-3 expressing the silase J1 gene was EcoRI
And XhoI (Takara Shuzo) to produce a 2.6 kb insert DNA fragment containing the structural gene of GL-7ACA acylase J1 and its own promoter and terminator, which were separated by agarose gel electrophoresis, Elute from the gel by electrophoresis, treat by phenol extraction and ethanol precipitation, and dissolve in TE buffer. On the other hand, Escherichia coli-B. Subtilis shuttle vector named pHY300PLK (Takara Shuzo) (10 μg)
Was digested with EcoRI and SalI and the resulting 4.9 kb D
The NA fragment is purified as described above. pACYJ1-
3 purified EcoRI-XhoI digested fragment (1 μg) and pHY30
The 0 PLK EcoRI-SalI digested fragment (1 μg)
Ligation is carried out at 12 ° C. for 16 hours using 5 units of T4 DNA ligase. In this ligation mixture, E. Cori MC10
Transform 61. Transformants are selected on LM agar plates containing 35 μg / ml ampicillin (Fujisawa Pharmaceutical) and confirmed by insert size and the presence of GL-7ACA acylase activity. Plasmid DNA is extracted from one of the recombinant strains by the clarified lysate method and named pACYJ1-B1. This plasmid p
Bacillus subtilis IS with ACYJ1-B1 (1 μg)
Transform W1214 (Takara Shuzo). Transformation was performed by the method of Anagostopoulos et al. (C. Anagostopoulos and JS
pizizen, J .; Bacteriol. 81 , 741-746 (196
Refer to 1)). Transformants are selected on L agar containing 20 μg / ml tetracycline. B. carrying pACYJ1-B1 obtained as above. Sachiris IS
W1214 is cultured for 18 hours at 30 ° C. in L-broth containing 20 μg / ml tetracycline. The supernatant and cell pellet are prepared by centrifugation of the culture broth at 10,000 g for 20 minutes. The GL-7ACA acylase activity of the supernatant, cell pellet and whole culture is determined by the HPLC assay described in Example 1.3.
GL-7ACA of supernatant, cell pellet and whole culture
The acylase activity is 0.63, 0.04 and 0.70 units / g bacteria (wet weight), respectively. GL-7ACA
Approximately 90% of the activity is recovered from the supernatant. In addition, the original B. Laterosporous J
No GL-7ACA acylase activity was detected in the whole culture of one strain, and the GL-7ACA acylase activity could be detected only when the cells were cultured on agar cells.

Example 4 B. Laterosporous J1 GL-7ACA acylase
Purification

4.1. Acylase from recombinant Escherichia coli
Purified 1% trypton (Dif Laboratories, USA)
An aqueous medium (L broth) (200 ml) consisting of 5% yeast extract (Difco) and 0.5% NaCl was placed in each of five 500 ml flasks, sterilized by an autoclave at 121 ° C. for 20 minutes, and separately sterilized by filtration. Add 20 μg / ml kanamycin sulfate. PACYJ1-
E.1 having One platinum loop of slant culture of E. coli JM109 is inoculated, and the microorganism is cultured at 30 ° C. for 24 hours while shaking at 300 rpm on a rotary shaker. On the other hand, an aqueous medium (18 l) containing the same components as above plus 0.04% adecanol (Asahi Denka) was placed in a 30 l jar fermenter, sterilized at 121 ° C. for 15 minutes, and separately sterilized by filtration. Add 0.25 mg / ml IPTG. After inoculating the medium with the entire amount of the culture broth obtained above, the microorganism is cultured at 30 ° C. for 18 hours. After the culture, 8000
Collect cells by continuous flow centrifugation at rpm. Approximately 80 g of the obtained cell pellet was added to a 0.1 M phosphate buffer (p
H8) suspended in 320 ml and disrupted by sonication in an ice-water bath for 5 minutes. The sonicated suspension is centrifuged at 8000 g for 10 minutes, and to the resulting supernatant is added Polymin P (Bethesda Research Laboratories, USA) to a final concentration of 0.3%. After stirring at 4 ° C. for 30 minutes, the mixture is centrifuged at 8000 g for 20 minutes. 2 for the supernatant
Dialyze against 0 mM Tris-HCl (pH 8.5). The crude extract was applied to a column of DEAE-Toyopearl 650M (Tosoh) equilibrated with 20 mM Tris-HCl buffer (pH 8.5).
(Layer volume: 100 ml). After washing the column with the same buffer, a 20 mM Tris-HCl buffer (pH 8.5) 300
Elution is carried out with a linear gradient of NaCl in ml (0-0.5 M). Fractions containing GL-7ACA activity are pooled and dialyzed against 20 mM Tris-HCl buffer (pH 8.5). The dialysate is applied to a column (layer volume: 30 ml) of DEAE-Toyopearl 650S (Tosoh) equilibrated with the same buffer. After washing the column with the same buffer, elution is carried out with a linear gradient (0-0.35 M) of NaCl in 100 ml of 20 mM Tris-HCl buffer (pH 8.5). Active fractions are pooled and dialyzed against 50 mM phosphate buffer (pH 8.0). The dialysate was applied to a column of HCA100S (Mitsui Toatsu) equilibrated with 50 mM phosphate buffer (pH 8.0) (layer volume:
20 ml). After washing the column with the same buffer,
Linear concentration gradient of phosphate buffer (pH 8.0) (0.05 to
Elution is carried out using 0.4M) (60 ml). Active fractions are pooled and dialyzed against 0.1 M phosphate buffer (pH 8.0). Ammonium sulphate in the dialysate, 35% final concentration
And butyl-Toyopearl 650 equilibrated with the same buffer containing 35% saturated ammonium sulfate.
Apply to a column of S (Tosoh) (layer volume: 20 ml). After washing the column with the same buffer, 0.1 M phosphate buffer (pH
8.0) in a linear concentration gradient of ammonium sulfate (35-0).
%). Active fractions are pooled and 20m
Dialyze against M Tris-HCl (pH 8.5). The dialysate is applied to a DEAE-G (Waters, USA) HPLC column (0.82 x 7.5 cm) equilibrated with the same buffer. Elution is carried out at a flow rate of 3 ml / min for 30 minutes using a linear gradient of NaCl (0-0.5 M) in 20 mM Tris-HCl (pH 8.5). Fractions containing GL-7ACA acylase activity were pooled and filtered on a UF-10PS filter.
Concentrate by ultrafiltration using (Tosoh). The total amount of the purified enzyme preparation is 9.6 mg, and its purity is estimated to be 95%.

4.2. From recombinant Bacillus subtilis
Purification of Acylase An aqueous medium (L broth) (180 ml) consisting of 1% tryptone (Difco Laboratories), 0.5% yeast extract (Difco) and 0.5% NaCl was placed in each of two 500 ml flasks, The solution is sterilized by an autoclave at 121 ° C. for 20 minutes, and 20 μg / ml tetracycline, which is separately sterilized by filtration, is added. In these mediums, B.A. One loopful of loop culture of S. subtilis ISW1214 was inoculated, and the microorganism was shaken at 30 ° C.
For 24 hours. On the other hand, L broth (1.8 L) is added to each of two 5 L flasks, sterilized at 121 ° C. for 20 minutes, and tetracycline is added. In each of these media,
After inoculating the whole amount of the culture broth obtained above, the microorganism was shaken at 30 ° C. for 6 hours at 300 rpm on a rotary shaker.
Culture until the optical density at 00 nm is 2.5.
The culture broth is then centrifuged at 10000 g for 20 minutes to remove the cell pellet and the resulting supernatant (3.
6l) is dialyzed against tap water and then against 20 mM Tris-HCl buffer (pH 8). Dialysate to 20 mM
The column is loaded on a DEAE-Toyopearl 650M column (layer volume: 100 ml) equilibrated with Tris-HCl (pH 8). After washing the column with the same buffer, 200 mM NaCl-20m
Elute with M Tris-HCl buffer (pH 8). The active fractions were pooled, adjusted to 60% saturation with ammonium sulfate,
Mix for 1 minute and centrifuge at 10000 g for 20 minutes. The resulting supernatant is further adjusted to 80% saturation with ammonium sulfate, stirred and centrifuged as above. The pellets obtained are mixed with 5% ammonium sulfate containing 45% of saturation.
Dissolve in 0 mM phosphate buffer (pH 8) and add Toyopearl HW5
The mixture is applied to a column (layer volume: 30 ml) of 5F (Tosoh). 50m
Elution is carried out with 35% saturated ammonium sulfate in M phosphate buffer (pH 8). Active fractions are pooled and dialyzed against 50 mM phosphate buffer (pH 8). Dialysate, TS
HPLC column of K gel G2000SW (Tosoh) (2.
15 × 60 cm). 500 mM phosphate buffer (pH
Perform elution in 8). The active fractions were pooled and dialyzed against 20 mM Tris-HCl (pH 8) to give DEAE-Toyopearl 6
Apply to a 50 M column (layer volume: 1 ml). 200 mM N
Elution is carried out with aCl-20 mM Tris-HCl (pH 8). Active fractions are pooled and dialyzed against 50 mM Tris-HCl (pH 8). The total amount of the final enzyme preparation is 1mg and its purity is 93%
It is estimated to be.

Example 5 B. from recombinant E. coli Laterosporous J1 GL-
Characterization of 7ACA acylase

5.1. Specific enzyme activity The activity of GL-7ACA acylase J1 is determined by HPLC assay. (a) 100 mM glycine buffer (pH
9) 1.4-20 mM GL-7ACA and 11 μg
(B) 100 mM Tris-HCl buffer (pH 8),
A reaction mixture (200 μl) containing 0.16-5 mM GGL-7ACA and 5 μg of enzyme is used. The reaction mixture is incubated at 37 ° C. for 2 minutes and 200 μl of 4% acetic acid
To stop the reaction. The HPLC conditions are the same as those described in Example 1.3. Specific enzyme activity is expressed as units per mg of protein. The protein concentration is determined by a Bio-Rad protein assay kit (Bio-Rad, USA) using bovine serum albumin as a standard.
Maximum specific enzyme activity (Vmax) and Michaelis constant (Km)
Calculated from slopes and intercepts obtained by a Lineweaver-Burk plot (see M. Dixon and EC Webb, Enzyme, Longman, London, 1958). The maximum specific enzyme activity of GL-7ACA acylase J1 in glycine buffer (pH 9) is 5.3 units / mg and that in Tris-HCl buffer (pH 8) is 4.8 units / mg. Glycine buffer (pH
The Km value of GL-7ACA acylase J1 in 9) was 3.2.
mM, 0.14 mM in Tris-HCl buffer (pH 8)
M.

5.2. Enzyme activity is determined by a substrate profile HPLC assay. 20 μmol of Tris-HCl buffer (pH 8), 2 mg of substrate and 22
A reaction mixture (200 μl) containing μg of enzyme is used.
The reaction mixture was incubated at 37 ° C. for 2 minutes,
The reaction is stopped by adding% acetic acid. GL relative enzyme activity
Expressed as a percentage compared to the activity against -7ACA.
Table 4 shows the results.

[0064]

[Table 4]

5.3. Effect of temperature a) Optimum temperature (see FIG. 8) 20 μmol of Tris-HCl buffer (pH 8), 0.4 mg of G
A reaction mixture (200 μl) containing L-7ACA and 22 μg of enzyme is used. The reaction is carried out at various temperatures between 25 and 50 ° C. for 2 minutes. The optimum temperature is 40 ° C.

B) Thermostable 23 μg / ml partially purified GL-7ACA acylase J1
(Purity 16%) in 0.1 M phosphate buffer (pH 8) at 45 ° C
For 2 hours, the residual enzyme activity was measured using 0.1 M Tris-HCl buffer (pH 8), 2 mg / ml GL-7ACA and 2.
Assay in a reaction mixture containing 3 μg / ml of treated enzyme. The reaction is performed at 37 ° C. for 2 minutes.

5.4. Influence of pH a) Optimum pH (see FIG. 9) 20 μmol of buffer (Macilvain buffer for pH 6, phosphate buffer for pH 6-8, Tris-HCl buffer for pH 7-9, pH 8-9 Uses glycine buffer)
A reaction mixture (200 μl) containing 0.4 mg GL-7ACA and 22 μg enzyme is used. Reaction is 37 ° C
For 10 minutes. The optimal pH for GL-7ACA acylase J1 is 8.

B) pH profile of thermostability (see FIG. 10) 100 μg / ml of GL-7ACA acylase J1 are treated for 1 hour at 50 ° C. in 0.1 M buffer at various pHs (p.
A phosphate buffer was used for H6.7 and H8, and a Tris-HCl buffer was used for pH9). 20 μmol glycine buffer (pH
8) Assay residual enzyme activity in a reaction mixture (200 μl) containing 0.4 mg GL-7ACA and 2 μg of treated enzyme. The reaction is performed at 37 ° C. for 10 minutes.

5.5. Inhibition by reaction products a) For the enzyme activity of GL-7ACA acylase J1
Reaction products on the enzyme activity of the influence GL-7ACA acylase J1 of the reaction product, i.e., to consider the 7ACA and inhibitory activity of glutaric acid. Enzyme activity is assayed in the presence of various concentrations of 7-ACA or glutaric acid. 7-ACA K
To determine i, 20 μmol of glycine buffer (pH 9),
0.39-2.0 μmol GL-7ACA, 0.3-
The reaction is performed at 37 ° C. for 6 minutes using a reaction mixture (200 μl) containing 0.48 μmol 7-ACA and 11 μg enzyme. To determine the Ki of glutaric acid, (a) 20 μ
Molar glycine buffer (pH 9), 0.26-3.0 μmol GL-7ACA, 2-10 μmol glutaric acid and 11 μg enzyme or (b) 20 μmol Tris-HCl buffer (pH 8). 05-1.0 μmol GL-7AC
A, 0.25-1.0 μmol glutaric acid and 5 μg
A reaction mixture (200 μl) containing the following enzymes is used. The reaction is performed at 37 ° C. for 3 minutes. Lineweaver-Burk plots in the absence or presence of both inhibitors indicate that they have the same vertical intercept, indicating that the mode of inhibition by both inhibitors is competitive. Apparent Michaelis constant
A Ki value is calculated from (Kmapp), Km and Vmax. 7-
The Ki value of ACA is 2.0 mM. The Ki value of glutaric acid in the reaction using 0.1 M glycine buffer (pH 9) was 53.
At mM, it is 0.99 mM in a reaction with 0.1 M Tris-HCl buffer (pH 8).

B) Effect of various enzyme inhibitors: p-chloromercuric benzoate (pCMB, Sigma Chemical), phenylmethylsulfonyl fluoride (PMSF, Sigma) and ethylenediaminetetraacetic acid (EDTA, Hanoi Tesque) were GL The effect on the activity of -7ACA acylase J1 is examined as follows. GL-7ACA acylase J
11 μg to 200 μl of 0.1 M Tris-HCl buffer
1.0 or 5.0 mM pCMB, (pH 8)
Or 5.0 mM PMSF or 1.0 or 5.
Treat with 0 mM EDTA at 37 ° C. for 3 hours. The remaining activity of the treated enzyme was determined by adding GL-7A as a substrate to the mixture.
It is determined by adding 22 μl of CA (20 mg / ml). The reaction is performed at 37 ° C. for 10 minutes. The residual activity is expressed as a percentage compared to the activity of the enzyme treated with the blank solution. Table 5 shows the results.

[Table 5]

5.6. Determination of isoelectric point (pI) Analytical isoelectric focusing of GL-7ACA acylase J1 was performed by the method of Olson et al. (B. Olsson, CE Nord,
T. Wadstrom and B.A. Wretlind, FEMS Microb
iol. Lett. 1 , 157-162 (1977)). Formulations of GL-7ACA acylase J1 purified from recombinant strains of Escherichia coli and Bacillus subtilis are applied to a thin layer of polyacrylamide gel containing 2% ampholine pH range 3.5-10 (Pharmacia LKB Biotechnology, Sweden). Isoelectric focusing SJ
Using -1071E (Atto), the protein was treated at 100V for 2 hours.
Perform time isoelectric focusing. After electrophoresis, the gel was stained with Coomassie Brilliant Blue-R-250,
The isoelectric point is determined from a calibration curve created by running a marker (Pharmacia LKB Biotechnology). The isoelectric point of the GL-7ACA acylase J1 standard purified from recombinant strains of Escherichia coli and Bacillus subtilis is estimated to be 4.7, which is the same.

5.7. SDS-polyacrylamide gel
Determination of molecular weight by electrophoresis Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis was performed by the method described in Laemli (UK Laemml).
i: Nature 237 , 680-685 (1970)). Egg white lysozyme (molecular weight 14,000), soybean trypsin inhibitor (21,000), bovine carbonic anhydrase (31,000), egg white ovalbumin (4
(3,000), bovine serum albumin (68,000), rabbit muscle phosphorylase b (97,000) are purchased from Bio-Rad Laboratories and used as molecular weight standards.
GL-7 purified from recombinant Escherichia coli or Bacillus subtilis
The final preparation of ACA acylase J1 shows one homogeneous band that is clearly distinguished by SDS-gel electrophoresis. Both G
The molecular weight of the L-7ACA acylase J1 preparation is the same,
Calculated as 0000.

5.8. Determination of amino acid sequence GL-7A purified from recombinant strains of Escherichia coli and Bacillus subtilis
A sample of CA acylase J1 was placed in 8 M urea at 37 ° C. for 30 minutes.
Denature by incubating for minutes and subject to reverse phase HPLC. The column used is Cosmosil 5C4-300.
(4.6 mm x 15 cm, Hanoi Tesque). Elution is 0.
Linear gradient of acetonitrile in 1% trifluoroacetic acid
(30% -60%) at a flow rate of 1 ml / min for 20 minutes. The amino acid sequences of the two J1 acylase preparations are
Determined by Gas Phase Sequencer 470A (Applied Biosystems, USA). The N-terminal amino acid sequences of both preparations are identical, and Gln-Ser-Glu-Gln-Glu-Lys-
Ala-Glu-Glu.

The newly isolated B. Laterosporous J
1 and the following transformants into which the expression plasmids obtained in the above Examples were inserted were one of the international depositary institutions according to the Budapest Treaty, 1-chome, Higashi 1-chome, Tsukuba, 305
3. Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology
Deposited on March 18.

Microorganism deposit number Bacillus Laterosporus J1 FERM BP-3106 Escherichia coli JM109 (pACYJ1-3) FERM BP-3105 Bacillus subtilis ISW1214 (pACYJ1-B1) FERM BP-3107

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction map of a plasmid pACYJ1-1.

FIG. 2 shows the construction of plasmid pACYJ1-3 and a restriction map.

FIG. 3 shows the construction and restriction cleavage map of plasmid pACYJ1-B1.

FIG. 4 shows the nucleotide sequence of GL-7ACA acylase J1 chromosome gene and the amino acid sequence deduced therefrom.

FIG. 5 is a view showing a nucleotide sequence of a GL-7ACA acylase J1 chromosome gene and an amino acid sequence deduced therefrom.

FIG. 6 shows the nucleotide sequence of the GL-7ACA acylase J1 chromosome gene and the amino acid sequence deduced therefrom.

FIG. 7 shows the nucleotide sequence of the GL-7ACA acylase J1 chromosome gene and the amino acid sequence deduced therefrom.

FIG. 8 is a graph showing the optimum temperature of GL-7ACA acylase J1.

FIG. 9 is a graph showing the optimum pH of GL-7ACA acylase J1.

FIG. 10. Stability of GL-7ACA acylase J1
It is a figure which shows a pH profile.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:19) (C12N 9/80 C12R 1:19) (C12N 9/80 C12R 1:07) (C12N 15/09 ZNA C12R 1:07) (72) Inventor Mana Tsumura 2-14, Azuma, Tsukuba City, Ibaraki Prefecture 912-304 (72) Inventor Seita Iwami 2-8-1 Umezono, Tsukuba City, Ibaraki Prefecture (72) Inventor Jin Takanari Ibaraki Prefecture 1160-7 Eikuni, Tsuchiura-shi (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 1/00-1/38 C12N 9/00-9/90 C12P 1/00 -41/00 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (9)

(57) [Claims] 1. A GL-7ACA acylase having the following properties: (a) having the ability to enzymatically convert glutaryl 7-ACA, adipyl 7-ACA and succinyl 7-ACA to 7-aminocephalosporanic acid; (b) molecular weight 70,000 daltons (SDS-PAGE)
(C) the N-terminal amino acid sequence is Gln-Ser-Glu-
Gln-Glu-Lys-Ala-Glu-Glu-. 2. It comprises the amino acid sequence of amino acid No. 1-607 shown in FIG. 4, FIG. 5, FIG. 6 and FIG. 7 or an amino acid sequence in which amino acids are deleted, substituted or added in said amino acid sequence, and GL-7ACA acylase having enzymatic activity to enzymatically convert ACA, adipyl 7-ACA and succinyl 7-ACA to 7-aminocephalosporanic acid. 3. GL-7ACA according to claim 1 or 2.
DNA encoding 4. The DNA according to claim 3, having the nucleotide sequence shown in FIG. 4, FIG. 5, FIG. 6, and FIG. 5. An expression vector comprising the DNA according to claim 3 or 4. 6. A host cell transformed with the expression vector according to claim 5. 7. The host cell according to claim 6, which is Escherichia coli or Bacillus subtilis. 8. A method for producing GL-7ACA acylase, comprising culturing the host cell according to claim 6 or 7 in a medium, and collecting GL-7ACA acylase from the culture. 9. A compound of the formula (II) Wherein R ¹ is acetoxy, hydroxy or hydrogen;
² represents carboxy (C1-C6) alkanoyl or D-glutamyl, respectively) or a salt thereof with a culture of the transformant according to claim 6 or 7 or a treated product thereof. The expression
(I) (Wherein R ¹ has the same meaning as described above) or a salt thereof.